The phosphorylation of protein has been proposed as one of the molecular mechanisms involved in cellular regulation. The protein kinases catalyze these phosphorylation reactions. In this investigation, properties of protein kinase activity associated with cell cytoskeletal proteins have been studied in both neuronal and nonneuronal tissues. In the squid giant axon the kinase activity associated with neurofilaments phosphorylates the high molecular weight 300 and 220 kilodalton neurofilament proteins. The squid axon neurofilament-associated protein kinase (SANFPK) activity appears to be both cyclic-AMP and calcium independent, and can phosphorylate both casein and histone. The squid axon neurofilament protein kinase utilizes both ATP and GTP in the phosphotransferase reaction. In rat brain the kinase activity is associated with microtubule-associated proteins and phosphorylates them. This kinase is cyclic-AMP dependent and its activity increases with low concentrations (14-60 millimolar) of ethanol. In erythrocyte ghosts isolated from rat red blood cells, the kinase activity is associated with the red blood cell cytoskeletal (spectrin and actin) preparation and the kinase specifically phosphorylates spectrin. This kinase also appears to be both cyclic-AMP and calcium independent. Its activity increases in the presence of low concentrations (10-100 millimolar) of ethanol. The observation that ethanol affects protein kinase activity provides an experimental basis for investigating the effects of ethanol on the structure and function of these cytoskeletal proteins and enzymes.